Roughly 30% of Trypanosoma cruzi infections result in potentially fatal chronic pathology known as Chagas disease. Our long-term goal is to identify likely targets for Chagas disease prevention and intervention. Prerequisite to this goal is the discovery and characterization of genes and their pattern of expression in T. cruzi. The recent completion of sequencing and annotation of the T. cruzi genome provides an unprecedented opportunity to identify new targets for vaccine and therapeutic interventions. We hypothesize that: 1) stages in the T. cruzi life cycle each have unique signatures of gene expression, 2) determining these signatures will be instructive as to the nature of the mechanisms and signaling pathways involved in stage conversion, and 3) that genes expressed non-constitutively during the T. cruzi lifecycle will include many genes that will be particularly useful targets for disease prevention and intervention. The aims of this project are to initiate the development of a high quality, annotated gene expression database of T. cruzi trypomastigotes (TRP), amastigotes (AMA), epimastigotes (EPI), and metacyclic trypomastigotes (MET). We will determine gene expression signatures using RNAs from a single time point in each life-cycle stage to interrogate DNA micro-arrays containing long oligonucleotides complementary to every annotated gene in the newly sequenced T. cruzi genome. Both total KNA and polysomal RNAs will be used in this analysis in order to investigate the relative contributions of mRNA stability and translational efficiency to control of gene expression in T. cruzi. The results of these analyses will be compared with proteomic data generated in our laboratory to determine if profiling using DNA micro-arrays accurately reflects protein presence and abundance. We will also perform a time-course study of TRP-to- AMA stage conversion to examine the fluctuations hi gene expression during this critical process in parasite development. Of the five stage transitions in the T. cruzi life cycle, the TRP-to-AMA is the most amenable to time course experimentation and is particularly relevant to the host-parasite relationship in T. cruzi-infected hosts. Moreover, using polysomal RNA and extending the time course will build upon our previous study of this stage transition. The data and analysis of these studies, along with proteome data, will be made available to the research community by deposition in TcruziDB in a MIAME compliant format. Completion of this study will provide valuable data on the expression of genes in T. cruzi, especially the many that are "hypothetical," and provide a starting point for elucidating the functions of these genes.